Methods for preparing enriched human hematopoietic cell preparations

ABSTRACT

The present invention relates to an antibody composition which contains antibodies specific for glycophorin A, CD3, CD24, CD16, CD14, and optionally CD45RA, CD36, CD56, CD2, CD19, CD66a and/or CD66b. A process is also provided for enriching and recovering human hematopoietic progenitor cells and stem cells in a sample containing human hematopoietic differentiated, progenitor, and stem cells. The process involves reacting the sample with an antibody composition containing antibodies capable of binding to the antigens glycophorin A, CD3, CD24, CD16, and CD14, and optionally CD45RA, CD36, CD56, CD2, CD19, CD66a and/or CD66b under conditions so that cell conjugates are formed between the antibodies and differentiated cells having the antigens glycophorin A, CD3, CD24, CD16, and CD14, and optionally CD45RA, CD36, CD56, CD2, CD19, CD66a and/or CD66b on their surfaces. The cell conjugates are removed and a cell preparation is obtained which is enriched in human hematopoietic progenitor cells and stem cells. The invention also relates to kits for carrying out this process and to the cell preparations prepared by the process.

FIELD OF THE INVENTION

The present invention relates to novel antibody compositions, andprocesses and kits for preparing cell preparations containing humanhematopoietic cells, and the use of the cell preparations. The inventionalso relates to purified human hematopoietic cell preparations.

BACKGROUND OF THE INVENTION

Blood cells have a relatively short life span and need to be replenishedthroughout life. In adults, blood cell formation or hematopoiesis takesplace in the bone marrow, but blood-forming stem cells can also be foundin peripheral blood. Hematopoietic cells represent a hierarchy ofproliferating and differentiating cells. The most abundant are thedifferentiating or lineage committed cells. These cells have limited orno proliferative capacity and represent specialized end cells that arefound in blood, and their immediate precursors.

The immediate precursors of the differentiating cells are the progenitorcells. Most of these cells are restricted to differentiate along asingle lineage but they may have quite extensive proliferative capacity.Progenitor cells appear morphologically as blast cells, and theytypically do not have specific features of the hematopoietic lineage towhich they are committed.

Progenitor cells are derived from stem cells. Stem cells have beenhistorically defined by their ability to self-renew as well as togenerate daughter cells of any of the hematopoietic lineages. Thepresence of stem and progenitor cells may be detected by their abilityto produce colony-forming cells in culture, They may also be detected byscreening for the CD34 antigen which is a positive marker for earlyhematopoietic cells including colony forming cells and stern cells. Atpresent, the long term culture initiating cell (LTCIC) assay appears tobe the best way to detect stem cells, or at least the most primitiveprogenitor cells, using tissue culture methodologies.

There is a continued interest in developing stem cell purificationtechniques. Pure populations of stem cells will facilitate studies ofhematopoiesis. Transplantation of hematopoietic cells from peripheralblood and/or bone marrow is also increasingly used in combination withhigh-dose chemo- and/or radiotherapy for the treatment of a variety ofdisorders including malignant, nonmalignant and genetic disorders. Veryfew cells in such transplants are capable of long-term hematopoieticreconstitution, and thus there is a strong stimulus to developtechniques for purification of hematopoietic stem cells. Furthermore,serious complications and indeed the success of a transplant procedureis to a large degree dependent on the effectiveness of the proceduresthat are used for the removal of cells in the transplant that pose arisk to the transplant recipient. Such cells include T lymphocytes thatare responsible for graft versus host disease (GVHD) in allogenicgrafts, and tumour cells in autologous transplants that may causerecurrence of the malignant growth.

Hematopoietic cells have been separated on the basis of physicalcharacteristics such as density and on the basis of susceptibility tocertain pharmacological agents which kill cycling cells. The advent ofmonoclonal antibodies against cell surface antigens has greatly expandedthe potential to distinguish and separate distinct cell types. There aretwo basic approaches to separating cell populations from bone marrow andperipheral blood using monoclonal antibodies. They differ in whether itis the desired or undesired cells which are distinguished/labeled withthe antibody(s). In positive selection techniques the desired cells arelabeled with antibodies and removed from the remainingunlabeled/unwanted cells. In negative selection, the unwanted cells arelabeled and removed. Antibody/complement treatment and the use ofimmunotoxins are negative selection techniques, but FACS sorting andmost batch wise immunoadsorption techniques can be adapted to bothpositive and negative selection. In immunoadsorption techniques cellsare selected with monoclonal antibodies and preferentially bound to asurface which can be removed from the remainder of the cells e,g. columnof beads, flasks, magnetic particles. Immunoadsorption techniques havewon favour clinically and in research because they maintain the highspecificity of targeting cells with monoclonal antibodies, but unlikeFACSorting, they can be scaled up to deal directly with the largenumbers of cells in a clinical harvest and they avoid the dangers ofusing cytotoxic reagents such as immunotoxins, and complement.

Current positive selection techniques for the purification ofhematopoietic stem cells target and isolate cells which express CD34(approximately 1-2% of normal bone marrow) (Civin, C. I., Trischmann, T.M., Fackler, M. J., Bernstein, I. D., Buhring, H. J., Campos, L. et al.(1989) Report on the CD34 cluster workshop, In: Leucocyte typing IV,White Cell Differentiation Antigens. Knapp, W., Dorken, B., Gilks, W.R., Reiber, E. P., Schmidt, R. E., Stein, H., and Kr. von den Borne,AE.G Eds., Oxford University Press. Oxford, pp.818). Thus, the potentialenrichment of hematopoietic stem cells using this marker alone is 50fold. Available techniques typically recover 30-70% of the CD34⁺ cellsin the start suspension and produce an enriched suspension which is50-80% CD34⁺ (Ishizawa, L. et al., In: Hematopoietic Stem Cells: TheMulhouse Manual eds. Wunder, E., Sovalat, H. Henon, P., and Serke, S.AlphaMed Presa, Ohio pp171-182; Shpall, E. J., et al. (1994), J. ofClinical Oncology 12:28-36; Winslow, J. M., et al. (1994), Bone MarrowTransplantation 14:265-271; Thomas, T. E., (1994), Cancer Research,Therapy and Control 4(2):119-128). The positive selection proceduressuffer from many disadvantages including the presence of materials suchas antibodies and/or magnetic beads on the CD34⁺ cells, and damage tothe cells resulting from the removal of these materials.

Negative selection has been used to remove minor populations of cellsfrom clinical grafts. These cells are either T-cells or tumour cellsthat pose a risk to the transplant recipient. The efficiency of thesepurges varies with the technique and depends on the type and number ofantibodies used. Typically, the end product is very similar to the startsuspension, missing only the tumor cells or T-cells.

Transplants of purified stern cells without differentiated or lineagecommitted cells will give short and long-term hematopoietic support(Shpall, E. J., et al. (1994), J. of Clinical Oncology 12:28-36). Sincedifferentiated cells make up a vast majority of the cells in bone marrowand blood, depletion of these cells produces a much smaller cellsuspension. The number of cells in the final product and the degree ofenrichment of progenitor/stem cells will depend on the efficiency of theantibody targeting and the removal of labeled cells.

There are several studies that enrich for hematopoietic stem cells bydepleting lineage committed cells but all require a number of positiveor negative selection steps to achieve the desired degree of enrichment(50 fold). Early studies required prior density separation and extensiveincubations to remove adherent cells (Linch, D. C., and Nathan, D. G.(1984), Nature 312 20/27:775-777; Sieff, C. A., et al. (1985), Science230:1171-1173; Kannourakis, G. and Bol, S. (1987) Exp. Hematol15:1103-1108.). More recent techniques have not been simplified;involving density separation steps followed by two partial lineagedepletions (Winslow, J. M., et al. (1994), Bone Marrow Transplantation14:265-271) or a partial lineage depletion using panning or FACSfollowed finally by positive selection using FACS (Carlo-Stella et al.1994, Blood 84, 10 supple.:104a; Reading, C., et al. (1994), Blood 84,10supple.:399a). Most of these methods for lineage depletion lackeffective antibody combinations against myeloid cells, erythrocytesand/or B-cells.

U.S. Pat. No. 5,087,570 describes a process for preparing ahematopoietic cell composition using a combination of positive andnegative selection, The process relies on the use of antibody to theSca-1 antigen which is associated with murine clonogenic bone marrowprecursors of thymocytes and progeny T-cells. The Sca-1 antibody is notuseful in isolating human hematopoietic cells.

U.S. Pat. No. 5,137,809 describes a method and kit for identifying andanalyzing lineages and maturational stages in normal hematopoieticcells. The method uses a first monoclonal antibody labeled with afluorochrome to react with all leukocytes in a sample, and a secondmonoclonal antibody labeled with a second fluorochrome to react with asubpopulation of leukocytes.

SUMMARY OF THE INVENTION

The present inventors have developed an antibody composition for use inpreparing cell preparations enriched for human hematopoletic stem cellsand progenitor cells. The antibodies in the antibody composition arespecific for selected markers associated with differentiated cells. Inparticular, the present inventors have found using a negative selectiontechnique that an antibody composition containing antibodies specificfor glycophorin A, CD3, CD24, CD16, CD14 and optionally CD45RA, CD36,CD2, CD19, CD56, CD66a, and CD66b, gives a cell preparation highlyenriched for human hematopoietic and progenitor cells. Maximumenrichment of early progenitor and stem cells (CD34⁺, CD38⁻ cells) wasobserved when anti-CD45R and anti-CD36 were included in the antibodycomposition.

The present inventors have shown that the use of the antibodycomposition of the present invention in a negative selection technique,to prepare a cell preparation which is enriched for hematopoietic stemcells and progenitor cells offers many advantages over conventionaltechniques. The antibody composition applied in one step to a sample ofperipheral blood, bone marrow, cord blood or frozen bone marrow, resultsin a greater than 50% recovery of human hematopoietic progenitor/stemcells with approximately a 3 log depletion of differentiated cells. Thishigh level of enrichment obtained using the antibody composition of theinvention, does not require additional enrichments steps, which wouldresult in loss of, or damage to, progenitor and stem cells. The recoveryof CD34⁺ cells, CD34⁺ CD38⁻ cells, colony forming cells, and LTCIC, isalso much higher than with conventional multistep techniques.

The enrichment and recovery of human hematopoietic progenitor and stemcells using the antibody composition of the invention in a negativeselection technique has many advantages over conventional positiveselection techniques. As mentioned above, a highly enrichedprogenitor/stem cell preparation can be obtained using a single step.The human progenitor and stem cells obtained using the antibodycomposition of the invention are not labeled or coated with antibodiesor modified making them highly suitable for transplantation and othertherapeutic uses.

Broadly stated the present invention relates to an antibody compositioncomprising antibodies specific for glycophorin A, CD3, CD24, CD16, CD14,and optionally CD45RA, CD36, CD56, CD2, CD19, CD66a and/or CD66b.

The present invention also broadly contemplates a process for enrichingand recovering human hematopoietic progenitor cells and stem cells in asample containing human hematopoietic differentiated, progenitor, andstem cells comprising reacting the sample with an antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD3, CD24, CD16, and CD14, and optionally CD45RA, CD36, CD56, CD2, CD19,CD66a and/or CD66b under conditions so that cell conjugates are formedbetween the antibodies and cells in the sample having the antigensglycophorin A, CD3, CD24, CD16, and CD14, and optionally CD45RA, CD36,CD56, CD2, CD19, CD66a and/or CD66b on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched in humanhematopoietic progenitor cells and stem cells.

The present invention also relates to a kit useful in performing theprocess of the invention comprising antibodies specific for glycophorinA, CD3, CD24, CD16, CD14, and optionally CD45RA, CD36, CD56, CD2, CD19,CD66a and/or CD66b, and instructions for performing the process of theinvention. The invention further relates to cell preparations obtainedin accordance with the process of the invention. The invention stillfurther contemplates a method of using the antibody composition of theinvention in a negative selection method to recover a cell preparationwhich is enriched in human hematopoietic progenitor and stem cells.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, reference is made herein to various publications,which are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the accompanyingdrawings, in which:

FIG. 1 is a schematic representation of magnetic cell labeling usingtetrameric antibody complexes and colloidal dextran iron; and

FIG. 2A shows a Fluorescence Activated Cell Sorting (FACS) profile ofnormal bone marrow before progenitor enrichment using lineage depletionwhere the vertical axis shows staining with CY5-antiCD34 and thehorizontal axis phycoerythrin-antiCD38.

FIG. 2B shows a Fluorescence Activated Cell Sorting (FACS) profile ofnormal bone marrow after progenitor enrichment using lineage depletionwhere the vertical axis shows staining with CY5-antiCD34 and thehorizontal axis phycoerythrin-antiCD38.

DETAILED DESCRIPTION OF THE INVENTION I. Hematopoietic Cell Types

The term "differentiated cells" used herein refers to humanhematopoietic cells which have limited or no proliferative capacity.Differentiated cells represent specialized end cells that are found inblood, and their immediate precursors.

The term "progenitor cells" used herein refers to cells which are theimmediate precursors of the differentiating cells. Most of theprogenitor cells differentiate along a single lineage but they may havequite extensive proliferative capacity. Progenitor cells appearmorphologically as blast cells, and they typically do not have specificfeatures of the hematopoietic lineage to which they are committed.

The term "stem cells" used herein refers to the cells from whichprogenitor cells are derived. Stem cells are defined by their ability toself-renew as well as to generate daughter cells of any of thehematopoietic lineages. Stem cells with long term hematopoieticreconstituting ability can be distinguished by a number of physical andbiological properties from differentiated cells and progenitor cells(Hodgson, G. S. & Bradley, T. R., Nature, Vol. 281, pp. 381-382; Visseret al., J. Exp. Med., Vol. 59, pp. 1576-1590, 1984; Spangrude et al.,Science, Vol. 241, pp. 58-62, 1988; Szilvassy et al., Blood, Vol. 74,pp. 930-939, 1989; Ploemacher, R. E. & Brons, R. H. C., Exp. Hematol.,Vol. 17, pp. 263-266, 1989).

The presence of stem cells and progenitor cells in a cell preparationmay be detected by their ability to produce colony-forming cells inculture. They may also be detected by screening for the CD34 antigenwhich is a positive marker for early hematopoietic cells includingcolony forming cells and stem cells. Primitive hematopoietic stem cellswith long term hematopoietic reconstituting ability can be identified bydetermining the number of clonogenic cells present after 5 to 8 weeks inlong term cultures (Sutherland et al., Blood, Vol. 74, p. 1563, 1986;Udomsakdi et al., Exp. Hematol., Vol. 19, p. 338, 1991; and, Sutherlandet al., Proc. Natl. Acad. Sci., Vol. 87, p. 3584, 1990).

II. Antibody Composition

As hereinbefore mentioned, the invention relates to an antibodycomposition comprising antibodies specific for the antigens glycophorinA, CD3, CD24, CD16, CD14, and optionally CD45RA, CD36, CD56, CD2, CD19,CD66a and/or CD66b which are present on the surface of humandifferentiated cells.

In an embodiment of the invention, an antibody composition is providedfor enriching and recovering human hematopoietic progenitor and stemcells from fresh bone marrow consisting essentially of antibodiesspecific for glycophorin A, CD3, CD24, CD16, CD14, CD66a and CD66b. In asecond embodiment, an antibody composition is provided for enriching andrecovering human hematopoietic progenitor and stem cells from previouslyfrozen bone marrow consisting essentially of antibodies specific forglycophorin A, CD3, CD24, CD16, and CD14. In a further embodiment of theinvention, an antibody composition is provided for enriching andrecovering human hematopoietic progenitor and stem cells from peripheralor cord blood consisting essentially of antibodies specific forglycophorin A, CD3, CD24, CD16, CD14, CD66a, CD66b, CD56 and CD2.

Pluripotent stem cells and committed progenitors express CD34, and thisCD34 compartment can be subdivided using antibodies to a variety of cellsurface markers. Stem cells co-purify in a population of CD34⁺ cellswhich lack or have low expression of certain lineage markers (CD38,CD33, CD45RA, CD71, CD36 and HLA-DR) (Craig et al. 1994, British Journalof Haematology, 88:24-30; Lansdorp, P. AI. and Dragowska, W. (1992) J.Exp. Med. 175:1501-1509; Sutherland, H, J., et al. (1989) Blood74.1563-1570). Antibodies recognizing these antigens can be included inthe antibody composition to further enrich for stem cells, while losingsome of the committed mature CD34⁺ cells. Preferably, anti-CD45RA andanti-CD36 are included in the antibody composition.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies and polyclonal antibodies, antibodyfragments (e.g., Fab, and F(ab')₂) and recombinantly produced bindingpartners. Antibodies are understood to be reactive against a selectedantigen on the surface of a differentiated cell if they bind with anaffinity (association constant) of greater than or equal to 10⁷ M⁻¹.

Polyclonal antibodies against selected antigens on the surface ofdifferentiated cells may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbits, mice, or rats.

Preferably, monoclonal antibodies are used in the antibody compositionsof the invention. Monoclonal antibodies specific for selected antigenson the surface of differentiated cells may be readily generated usingconventional techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614,4,543,439, and 4,411,993 which are incorporated herein by reference; seealso Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, andAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988, which are also incorporated herein byreference).

Other techniques may also be utilized to construct monoclonal antibodies(see William D. Huse et al., "Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda," Science246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library," Proc Natl. Acad. Sci USA 86:5728-5732,August 1989; see also Michelle Alting-Mees et al., "Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas," Strategies inMolecular Biology 3:1-9, January 1990; these references describe acommercial system available from Stratacyte, La Jolla, Calif., whichenables the production of antibodies through recombinant techniques).

Similarly, binding partners may be constructed utilizing recombinant DNAtechniques. Within one embodiment, the genes which encode the variableregion from a hybridoma producing a monoclonal antibody of interest areamplified using nucleotide primers for the variable region. Theseprimers may be synthesized by one of ordinary skill in the art, or maybe purchased from commercially available sources. The primers may beutilized to amplify heavy or light chain variable regions, which maythen be inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L(Stratacyte), respectively. These vectors may then be introduced into E.coli for expression. Utilizing these techniques, large amounts of asingle-chain protein containing a fusion of the V_(H) and V_(L) domainsmay be produced (See Bird et al., Science 242:423-426, 1988). Inaddition, such techniques may be utilized to change a "murine" antibodyto a "human" antibody, without altering the binding specificity of theantibody.

Antibodies against selected antigens on the surface of differentiatedcells may also be obtained from commercial sources.

Antibodies may be selected for use in the antibody compositions of theinvention based on their ability to deplete targeted differentiatedcells and recover non-targeted cells (i.e. progenitor and stem cells, orspecific differentiated cells) in magnetic cell separations as moreparticularly described herein, and in co-pending U.S. Pat. No.5,514,390, which is incorporated in its entirety herein by reference. Ingeneral, an antibody is selected that gives approximately a 3 logdepletion of differentiated cells, with greater than 75% recovery ofCD34⁺ cells (bone marrow, mobilized blood and cord blood) ornon-targeted lymphocytes (steady state blood), in test magnetic cellseparations as described herein.

The anti-glycophorin A antibodies contained in the antibody compositionof the invention are used to label erythrocytes. Examples of monoclonalantibodies specific for glycophorin A are 10F7MN (U.S. Pat. No.4,752,582, Cell lines: ATCC accession numbers HB-8473, HB-8474, andHB-8476), and D2.10 (Immunotech, Marseille, France). The concentrationof antiglycophorin A antibodies used in the antibody composition aregenerally less than the concentration that will cause agglutination(i.e. 3-10 μg/ml). Preferably the concentration of antiglycophorin Aantibodies used in the antibody composition is between about 0.5 to 5μg/ml, preferably 1 to 2 μg/ml.

Monoclonal antibodies against CD24, CD3, CD19, CD20, CD56, CD2 in theantibody composition of the invention are used to label B and Tlymphocytes and NK cells. Examples of monoclonal antibodies specific forCD24, CD3, CD19, CD20, CD56, and CD2, are 32D12 (Dr. Steinar Funderud,Institute for Cancer Research, Dept. of Immunology, Oslo, Norway,) andALB9 (Immunotech, Marseille, France); UCHT1 (Immunotech, Marseille,France) and Leu-4 (Becton Dickinson, Mountain View, Calif.); J4.119(Immunotech, Marseille, France) and Leu-12 (Becton Dickinson, MountainView, Calif.); MEM97 (Dr. Horejsi, Institute of Molecular GeneticsAcademy of Sciences of the Czech Republic, Praha, Czech Republic, orCedarlane Laboratories, Hornby, Ontario, Canada) and Leu-16 (BectonDickinson, Mountain View, Calif.); T199 (Immunotech, Marseille, France);and 6F10.3 (Immunotech, Marseille, France), respectively. Theconcentration of each of the monoclonal antibodies against CD24, CD3,CD19, CD20, CD56, CD2 contained in the antibody composition is betweenabout 0.5 to 5 μg/ml, preferably 2 to 3 μg/ml.

Monoclonal antibodies against CD14, CD16, CD66a and CD66b in theantibody compositions of the invention are used to label monocytes.Examples of monoclonal antibodies specific for CD14, CD16, CD66a andCD66b, are MEM15 and MEM18 (Dr. Vaclav Horejsi, Institute of MolecularGenetics Academy of Sciences of the Czech Republic, Praha, CzechRepublic; Cedarlane Laboratories, Hornby, Ontario, Canada); MEM154 (Dr.Vaclav Horejsi, Institute of Molecular Genetics Academy of Sciences ofthe Czech Republic, Praha, Czech Republic; Cedarlane Laboratories,Hornby, Ontario, Canada), Leu-11a (Becton Dickinson, Mountain View,Calif.), and 3G8 (Immunotech, Marseille, France); CLB/gran10 (CLB,Central Laboratory of the Netherlands, Red Cross Blood TransfusionService); and, B13.9 (CLB, Central Laboratory of the Netherlands, RedCross Blood Transfusion Service) and 80H3 (Immunotech, Marseille,France), respectively. The concentration of each of the monoclonalantibodies against CD14, CD16, CD66a and CD66b contained in the antibodycomposition is between about 0.5 to 5 μg/ml, preferably 2-3 μg/ml.

Monoclonal antibodies against CD45RA and CD36 are used to label T-cells,B-cells, granulocytes, platelets, monocytes, differentiated erythroidprecursors, and some committed mature progenitors, to further enrich forstem cells. Examples of monoclonal antibodies against CD45RA and CD36are 8D2.2 (StemCell Technologies, Vancouver, Canada, Craig et al., 1994,British Journal of Haematology, 88:24-30.), Leu-18 (Becton Dickinson,Mountain View, Calif.); and, FA60152 (Immunotech, Marseille, France) andIVC7 (CLB, Central Laboratory of the Netherlands Red Cross BloodTransfusion Service), respectively. The concentration of each of themonoclonal antibodies against CD45R and CD36 contained in the antibodycomposition is between about 0.5 to 5 μg/ml, preferably 1 to 3 μg/ml.

Table 2 sets out the most preferred monoclonal antibodies, their sourcesand concentrations, for use in the antibody compositions of theinvention.

In most preferred embodiments of the invention the antibody compositioncomprises the monoclonal antibodies designated D2.10, UCHT1, MEM15, 3G8,ALB9, 80H3, J4.119, 6F10.3, T199, 8D2.2 and FA60152 or comprises themonoclonal antibodies designated 10F7MN, UCHT1, 32D12, MEM154, MEM15,80H3 or B13.9, T199, 6F10.3, J4.119, and optionally, 8D2.2 and 1VC7.

Antibody compositions in accordance with the present invention may beprepared which lack antibodies to a specific differentiated cell type orlineage committed cell. For example, an antibody composition may beprepared which does not contain antibodies to the CD14, CD16, CD66a andCD66b antigens which are specific to monocytes. This composition may beused to prepare a cell preparation which is enriched for monocytes.Other examples of antibody compositions which can be used to preparecell populations enriched for monocytes, B-cells, T-cells, CD4⁺ T-cells,CD8⁺ T-cells, and NK cells are set out in Table 3.

III. Process for Preparing Cell Preparations Enriched in Progenitor/StemCells or a Differentiated Cell Type

The antibody composition of the invention may be used to enrich andrecover cell preparations enriched in human hematopoietic stem cells andprogenitor cells. In accordance with the process of the invention, asample is reacted with an antibody composition of the invention; undersuitable conditions, cell conjugates form between the antibodiescontained in the antibody composition which are specific for selectedantigens on the surface of differentiated cells, and the cells in thesample containing the antigens on their surface; and the cell conjugatesare removed to provide a cell preparation enriched in humanprogenitor/stem cells.

Conditions which permit the formation of cell conjugates may be selectedhaving regard to factors such as the nature and amounts of theantibodies in the antibody composition, and the estimated concentrationof targeted differentiated cells in the sample.

The antibodies in the antibody composition may be labelled with a markeror they may be conjugated to a matrix. Examples of markers are biotin,which can be removed by avidin bound to a support, and fluorochromes,e.g. fluorescein, which provide for separation using fluorescenceactivated sorters. Examples of matrices are magnetic beads, which allowfor direct magnetic separation (Kernshead 1992), panning surfaces e.g.plates, (Lebkowski, J. S., et al., (1994), J. of Cellular Biochemistrysupple. 18b:58), dense particles for density centrifugation (VanVlasselaer, P., Density Adjusted Cell Sorting (DACS), A Novel Method toRemove Tumor Cells From Peripheral Blood and Bone Marrow StemCellTransplants. (1995) 3rd International Symposium on Recent Advances inHematopoietic Stem Cell Transplantation-Clinical Progress, NewTechnologies and Gene Therapy, San Diego, Calif.), adsorption columns(Berenson et al. 1986, Journal of Immunological Methods 91:11-19.), andadsorption membranes (Norton et al. 1994). The antibodies may also bejoined to a cytotoxic agent such as complement or a cytotoxin, to lyseor kill the targeted differentiated cells.

The antibodies in the antibody composition may be directly or indirectlycoupled to a matrix. For example, the antibodies in the composition ofthe invention may be chemically bound to the surface of magneticparticles for example, using cyanogen bromide. When the magneticparticles are reacted with a sample, conjugates will form between themagnetic particles with bound antibodies specific for antigens on thesurfaces of the differentiated cells, and the differentiated cellshaving the antigens on their surfaces.

Alternatively, the antibodies may be indirectly conjugated to a matrixusing antibodies. For example, a matrix may be coated with a secondantibody having specificity for the antibodies in the antibodycomposition. By way of example, if the antibodies in the antibodycomposition are mouse IgG antibodies, the second antibody may be rabbitanti-mouse IgG.

The antibodies in the antibody composition may also be incorporated inantibody reagents which indirectly conjugate to a matrix. Examples ofantibody reagents are bispecific antibodies, tetrameric antibodycomplexes, and biotinylated antibodies.

Bispecific antibodies contain a variable region of an antibody in theantibody composition of the invention, and a variable region specificfor at least one antigen on the surface of a matrix. The bispecificantibodies may be prepared by forming hybrid hybridomas. The hybridhybridomas may be prepared using the procedures known in the art such asthose disclosed in Staerz & Bevan, (1986, PNAS (USA) 83:1453) and Staerz& Bevan, (1986, Immunology Today, 7:241). Bispecific antibodies may alsobe constructed by chemical means using procedures such as thosedescribed by Staerz et al., (1985, Nature, 314:628) and Perez et al.,(1985 Nature 316:354), or by expression of recombinant immunoglobulingene constructs.

A tetrameric immunological complex may be prepared by mixing a firstmonoclonal antibody which Is capable of binding to at least one antigenon the surface of a matrix, and a second monoclonal antibody from theantibody composition of the invention. The first and second monoclonalantibody are from a first animal species. The first and second antibodyare reacted with an about equimolar amount of monoclonal antibodies of asecond animal species which are directed against the Fc-fragments of theantibodies of the first animal species. The first and second antibodymay also be reacted with an about equimolar amount of the F(ab')₂fragments of monoclonal antibodies of a second animal species which aredirected against the Fc-fragments of the antibodies of the first animalspecies. (See U.S. Pat. No. 4,868,109 to Lansdorp, which is incorporatedherein by reference for a description of tetrameric antibody complexesand methods for preparing same).

The antibodies of the invention may be biotinylated and indirectlyconjugated to a matrix which is labelled with (strept) avidin. Forexample, biotinylated antibodies contained in the antibody compositionof the invention may be used in combination with magnetic iron-dextranparticles that are covalently labelled with (strept) avidin (Miltenyi S.et al., Cytometry 11:231, 1990). Many alternative indirect ways tospecifically cross-link the antibodies in the antibody composition andmatrices would also be apparent to those skilled in the art.

In an embodiment of the invention, the cell conjugates are removed bymagnetic separation using magnetic particles. Suitable magneticparticles include particles in ferrofluids and other colloidal magneticsolutions. "Ferrofluid" refers to a colloidal solution containingparticles consisting of a magnetic core, such as magnetite (Fe₃ O₄)coated or embedded in material that prevents the crystals frominteracting. Examples of such materials include proteins, such asferritin, polysaccharides, such as dextrans, or synthetic polymers suchas sulfonated polystyrene cross-inked with divinylbenzene. The coreportion is generally too small to hold a permanent magnetic field. Theferrofluids become magnetized when placed in a magnetic field. Examplesof ferrofluids and methods for preparing them are described by KemsheadJ. T. (1992) in J. Hematotherapy, 1:35-44, at pages 36 to 39, and Zioloet al. Science (1994) 257:219 which are incorporated herein byreference. Colloidal particles of dextran-iron complex are preferablyused in the process of the invention.(See Molday, R. S. and McKenzie, L.L. FEBS Lett. 170:232, 1984; Miltenyi et al., Cytometry 11:231, 1990;and Molday, R. S. and MacKenzie, D., J. Immunol. Methods 52:353, 1982;Thomas et al., J. Hematother. 2:297 (1993); and U.S. Pat. No. 4,452,733,which are each incorporated herein by reference).

FIG. 1 is a schematic representation of magnetic cell labeling usingtetrameric antibody complexes and colloidal dextran iron.

In accordance with the magnetic separation method, the sample containingthe progenitor and stem cells to be recovered, is reacted with the abovedescribed antibody reagents, preferably tetrameric antibody complexes,so that the antibody reagents bind to the targeted differentiated cellspresent in the sample to form cell conjugates of the targeteddifferentiated cells and the antibody reagents. The reaction conditionsare selected to provide the desired level of binding of the targeteddifferentiated cells and the antibody reagents. Preferably the sample isincubated with the antibody reagents for a period of 5 to 60 minutes ateither 4° or ambient room temperature. The concentration of the antibodyreagents is selected depending on the estimated concentration of thetargeted differentiated cells in the sample. Generally, theconcentration is between about 0.1 to 50 μg/ml of sample. The magneticparticles are then added and the mixture is incubated for a period ofabout 5 minutes to 30 minutes at the selected temperature. The sample isthen ready to be separated over a magnetic filter device. Preferably,the magnetic separation procedure is carried out using the magneticfilter and methods described in co-pending U.S. Pat. No. 5,514,340 toLansdorp and Thomas which is incorporated in its entirety herein byreference.

The sample containing the magnetically labelled cell conjugates ispassed through the magnetic filter in the presence of a magnetic field.In a preferred embodiment of the invention, the magnet is a solenoidelectromagnet with a 3" diameter bore and having a magnetic field of0.5-2 Tesla. The magnetically labelled cell conjugates are retained inthe high gradient magnetic column and the materials which are notmagnetically labelled flow through the column after washing with abuffer.

The preparation containing non-magnetically labelled cells may beanalyzed using procedures such as flow cytometry. The ability of thecells in the preparation to produce colony-forming cells or long termculture initiating cells (LTCIC) in culture may also be assessed. Theefficiency of the separation procedure may also be determined bymonitoring the recovery of CD34⁺ cells, CD34⁺ CD38⁻ cells and colonyforming cells.

The antibody compositions of the invention may also be used to prepare acell preparation which is enriched for a specific differentiated celltype. This is achieved by using antibody compositions which lackantibodies to the specific differentiated cell type, in the abovedescribed processes of the invention. Particular embodiments of theseprocesses of the invention are set out below. It will be appreciatedthat the markers, matrices, antibody reagents, and procedures describedherein may be used in these processes to facilitate recovery of cellpreparations enriched for a specific differentiated cell type. Examplesof antibodies which may be used in these processes are set out in Table2.

In accordance with one embodiment of the invention, a process isprovided for enriching and recovering monocytes from a blood or bonemarrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD2, CD3, CD56, and CD24 or CD19, and optionally CD66bunder conditions so that cell conjugates are formed between theantibodies and the cells in the sample having the antigens glycophorinA, CD2, CD3, CD56, and CD24 or CD19 and optionally CD66b, on theirsurfaces; removing the cell conjugates; and recovering a cellpreparation which is enriched in monocytes.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering B-cells from a blood or bonemarrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD2, CD3, CD56, and CD14, and optionally CD66b underconditions so that cell conjugates are formed between the antibodies andthe cells in the sample having the antigens glycophorin A, CD2, CD3,CD56, and CD14 and optionally CD66b, on their surfaces; removing thecell conjugates, and recovering a cell preparation which is enriched inB-cells.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering T-cells from a blood or bonemarrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD24 or CD19, CD56, and optionally CD66b under conditionsso that cell conjugates are formed between the antibodies and the cellsin the sample having the antigens glycophorin A, CD24 or CD19, CD56, andoptionally CD66b, on their surfaces; removing the cell conjugates; andrecovering a cell preparation which is enriched in T-cells.

In accordance with yet another embodiment of the invention, a process isprovided for enriching and recovering CD4⁺ T-cells from a blood or bonemarrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD24 or CD19, CD56, CD8, and optionally CD66b underconditions so that cell conjugates are formed between the antibodies andthe cells in the sample having the antigens glycophorin A, CD24 or CD19,CD56, CD8, and optionally CD66b, on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched in CD4⁺T-cells.

In accordance with a further embodiment of the invention, a process isprovided for enriching and recovering CD8⁺ T-cells from a blood or bonemarrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD24 or CD19, CD56, CD4, and optionally CD66b underconditions so that cell conjugates are formed between the antibodies andthe cells in the sample having the antigens glycophorin A, CD24 or CD19,CD56, CD4, and optionally CD66b, on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched in CD8⁺T-cells.

In accordance with a still further embodiment of the invention, aprocess is provided for enriching and recovering NK-cells from a bloodor bone marrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD24 or CD19, and CD3, and optionally CD66b underconditions so that cell conjugates are formed between the antibodies andthe cells in the sample having the antigens glycophorin A, CD24 or CD19,and CD3, and optionally CD66b, on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched inNK-cells.

IV. Uses of the Compositions and Processes of the Invention

The device and processes of the invention may be used in the processingof biological samples including blood in particular, cord blood andwhole blood. It has also been found that the antibody compositions ofthe invention can be used to prepare hematopoietic progenitor and stemcell preparations from bone marrow samples, including previously frozenbone marrow samples.

The processes of the invention are preferably used to deplete or purgeerythrocytes, B and T lymphocytes, monocytes, NK cells, andgranulocytes, from samples to prepare hematopoietic progenitor and stemcell preparations for use in transplantation as well as othertherapeutic methods that are readily apparent to those of skill in theart. For example, bone marrow or blood can be harvested from a donor inthe case of an allogenic transplant and enriched for progenitor and stemcells by the processes described herein.

Using the process of the invention it is possible to recover a highlypurified preparation of human hematopoietic stem/progenitor cells. Inparticular, a hematopoietic cell population containing greater than 50%of the hematopoietic progenitor/stem cells present in the originalsample, and which is depleted of differentiated cells in the originalsample by approximately 3 logarithms may be obtained. The humanhematopoietic progenitor and stem cells in the preparation are notcoated with antibodies, or modified making them highly suitable fortransplantation and other therapeutic uses that are readily apparent tothose skilled in the art.

The cell preparations obtained using the processes of the invention maybe used to isolate and evaluate factors associated with thedifferentiation and maturation of human hematopoietic cells. The cellpreparations may also be used to determine the effect of a substance oncell growth and/or differentiation into a particular lineage.

The antibody compositions and processes of the invention may also beused to prepare a cell preparation from samples such as blood and bonemarrow, which is enriched in a selected differentiated cell type. Thiswill enable studies of specific cell to cell interactions includinggrowth factor production and responses to growth factors. It will alsoallow molecular and biochemical analysis of specific cells types. Cellpreparations enriched in NK cells and T-cells may also be used in immunetherapy against certain malignancies.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Method for Evaluating Antibody Combinations

Suspensions of normal human bone marrow, human cord blood, mobilizedhuman peripheral blood and previously frozen human bone marrow werelabeled with tetrameric antibodies and colloidal dextran iron formagnetic cell depletions. Monoclonal antibodies recognizing lineagespecific cell surface antigens were mixed with a mouse IgG₁ anti-dextranantibody (Thomas, T. E., et al. (1992), J. Immunol Methods 154:245;252)and a rat IgG₁ monoclonal antibody which recognizes the Fc portion ofthe mouse IgG₁ molecule (TFL-P9) (Lansdorp, P. M., and Thomas, T. E.(1990), Mol. Immunol. 27:659-666). Tetrameric antibody complexes(Lansdorp, P. M., and Thomas, T. E. (1990), Mol. Immunol. 27;659-666;U.S. Pat. No. 4,868,109 to Lansdorp) spontaneously form when mouse IgG₁molecules (the lineage specific monoclonal antibody and anti-dextran)are mixed with P9. A proportion of these tetrameric antibody complexesare bifunctional, recognizing an antigen on the surface of the targetcell on one side and dextran (part of the magnetic colloidal dextraniron) on the other. Tetrameric antibody complexes were made for all theantibodies in the lineage cocktail. FIG. 1 shows a schematicrepresentation of magnetic cell labeling using tetrameric antibodycomplexes and colloidal dextran iron.

Cells were labeled for separation (1-5×10⁷ cells/ml) by incubating themwith the desired combination of tetramers for 30 min on ice followed bya 30 min incubation with colloidal dextran iron (final OD450=0.6)(Molday and MacKenzie 1982). The cells were then passed through amagnetic filter (U.S. Pat. No. 5,514,340; inventors Lansdorp and Thomas)at 1 cm/min. The magnetically labeled cells bind to the filter and theunlabeled cells pass through. FIG. 1 shows a schematic representation ofmagnetic cell labeling using tetrameric antibody complexes and colloidaldextran iron.

The flow through fraction is collected and analyzed for hematopoieticcolony forming cells (CFU-GM, CFU-C, LTCIC) (Eaves, C. J. and Eaves, A.J. (1992) In: Current Therapy in Hematology-Oncology, Fourth Edition pp.159-167), CD34+ cells, and CD34+CD38- cells. The enrichment of thesecell types depends on how well the antibody cocktail has targeted othercells for removal. Each antibody cocktail was evaluated for the purityand recovery of colony forming cells, CD34+ cells, and CD34+CD38- cells.FIG. 2 shows a FACS profile of normal bone marrow before and afterprogenitor enrichment via lineage depletion.

Example 2 Antibodies for the Enrichment of Progenitor Cells (ProgenitorCocktail)

The results of numerous cell separations identified a combination oflineage specific antibodies that produce the maximum enrichment andrecovery of CD34+ cells and colony forming cells.

Targeting Erythrocytes--Anti-glycophorin A antibodies were used to labelerythrocytes for depletion. Many of these antibodies will causeagglutination at moderate to high antibody concentrations (3-10 μg/ml).It was found that cells could be effectively targeted for magneticdepletion with concentrations of anti-glycophorin antibody that wereseveral fold lower than that which caused agglutination.

Targeting Lymphocytes--B, T, and NK cells were targeted with monoclonalantibodies against CD24, CD3, CD19, CD20, CD56, CD2. Initial depletionsof mobilized peripheral blood using just anti-CD24 and CD3 forlymphocyte depletion showed that a proportion of the CD34 negative cellsin the purified fraction were CD56 positive (NK cells). Subsequent testswith and without anti-CD56 increased the purity of CD34+ cells in therecovered fraction by 12-20%. Adding an anti-CD2 to the cocktailincreased the purity an additional 12-13%. Anti-CD2 and anti-CD56 had nosignificant effect on lineage depletions of fresh bone marrow. It islikely that bone marrow does not have as many CD3- CD2+ and CD3-CD56+cells. Anti-CD2 and anti-CD56 are added to the depletion cocktails formobilized peripheral blood but not bone marrow.

Antibodies against CD24 were sufficient to target all detectable B-cellsfor depletion. Adding anti-CD19 gave no additional enrichment of CD34+cells from mobilized peripheral blood or bone marrow. Substituting CD19for CD24 in separations of fresh bone marrow had no effect on theenrichment or recovery of C34+ cells, hematopoietic colony forming cellsand LTCIC, Replacing anti-CD24 with anti-CD20 or both anti-CD19 andanti-CD20 had no significant effect on separations of mobilizedperipheral blood. An effect was seen in a separation with cord blood;when anti-CD24 was replaced with anti-CD19, the purity was decreased21%, but recovery of CD34+ cells was increased 28%.

Targeting Mature Myeloid Cells--Monocytes were effectively targeted withan antibody against CD14 in all cell suspensions tested. The removal ofgranulocytes from peripheral blood and fresh bone marrow was moreefficient using both anti-CD16 and anti-CD66b rather than anti-CD16alone and adding anti-CD66a gave an additional 10% enrichment of CD34+cells from fresh bone marrow and 20% enrichment for peripheral blood.Anti-CD16 alone was sufficient to deplete the granulocytes frompreviously frozen marrow. Adding anti-CD41 or CD42a did not increase thepurity of CD34+ cells in either peripheral blood or bone marrow.

Example 3 Antibodies for the Enrichment of Stem Cells (Stem CellCocktail)

Both pluripotent stem cells and committed progenitors express CD34, butthe CD34 compartment can be further subdivided using a variety of cellsurface markers to isolate these cell types. Stem cells co-purify in apopulation of CD34+ cells which lack or have low expression of certainlineage markers (CD38, CD33, CD45RA, CD71, CD36 and HLA-DR) (Craig etal. 1994, British Journal of Haematology, 88:24-30; Lansdorp, F. AI. andDragowska, W. (1992), J. Exp. Med. 175:1501-1509; Sutherland, H. J., etal. (1989), Blood 74.1563-1570.). If antibodies recognizing theseantigens are included in the lineage cocktail one can further enrich forstem cells while losing some of the committed mature CD34+ cells.Antibodies to CD36 and CD45RA were added to the lineage cocktail tospecifically enrich for stem cells. The recovery of CD34+ CD38- cellsand LTCICs were monitored to determine the efficiency of the lineagedepletions with the "stem cell cocktail".

Including anti-CD45RA in the stem cell cocktail does not negate the needfor anti-CD24 in the cocktail nor does anti-CD36 allow the removal ofCD66a. Adding anti-CD36 did increase the purity of CD34+CD38-cells inseparations of previously frozen bone marrow by 15% . The addition ofanti-transferrin (CD71) antibody to the cocktail resulted in very poorrecovery of CD34+ CD38- cells, and LTCIC as well as producing asignificant number of dead cells in the enriched fraction (viabilitynormally >95%). Separations of previously frozen bone marrow with thestem cell cocktail produced a cell suspension which is approximately 30%CD34+ CD38- with 30% recovery of these cells, Similar separations withmobilized peripheral blood gave 15% purity and 72% recovery.

Example 4 Different Antibodies to the Same Antigen

The enrichment of hematopoietic progenitor cells via lineage depletionis not only dependent on the number of types of committed cells that aretargeted but also the effectiveness of this targeting and subsequentremoval using magnetic separation or other antibody mediated techniques.It was found that different antibodies recognizing the same antigen mayreproducibly produce different degrees of progenitor enrichment, Theanti-CD24 antibody 32D12 produced better results in lineage depletionsthan ALB9 (also anti-CD24); the purity of the enriched cell suspensionincreased 10% in a separation with cord blood. In cell depletions with asingle tetramer type, 32D12 out performed ALB9 and anti-glycophorinantibody 10P7MN out performed anti-glycophorin antibody D2.10 althoughswitching anti-glycophorin antibodies in a lineage depletion had nosignificant effect.

The criteria for choosing a particular antibody at a given concentrationis its performance in a magnetic cell separation which equates to themaximum depletion of antibody targeted cells with the maximum recoveryof non-target cells. Often depletion of antibody targeted cellsincreases with antibody concentration but so does the non-specificlabeling of cells. In general, the result looked for was a 3 logdepletion with greater than 75% recovery of CD34+ cells or non-targetedlymphocytes if the test cell suspension was steady state peripheralblood. The performance in a cell separation typically mimicked thedegree of specific cell labeling and the degree on non-specific labelingmeasured by FACS (sheep anti-mouse FITC staining). Staining experimentswere often run to eliminate antibodies (specific staining low,non-specific staining high) and reduce the number of antibodyconcentrations to be tested in cell separations.

While what is shown and described herein constitutes various preferredembodiments of the subject invention, it will be understood that variouschanges can be made to such embodiments without departing from thesubject invention, the scope of which is defined in the appended claims.

                  TABLE 1    ______________________________________    Optimal Antibody Cocktail for the Enrichment of Hematopoietic    Progenitors              optimum antibody                           % purity   % recovery    Cell Suspension              cocktail anti-                           CD34+ cells                                      CD34+ cells    ______________________________________    fresh bone              gly*, CD3, CD24,                           39,44,38   67,48,55    marrow    CD16, CD14,              CD66a, CD66b,    previously              gly, CD3, CD24,                           64,46,50,53                                      85,55,82,64    frozen bone              CD16, CD14,    marrow    mobilized gly, CD3, CD24,                           51,50,57   43,49,85    peripheral blood              CD16, CD14,              CD66a, CD66b,              CD56, CD2,    cord blood              gly, CD3, CD24,                           56,88,58,55,63                                      75,85,53,67,48              CD16, CD14,              CD66a, CD66b,              CD56, CD2,    ______________________________________     *gly = glycophorin A

                  TABLE 2    ______________________________________    Antibodies used in Lineage Depletions                                         Concen-                                         tration    Antigen Antibody  Source             ug/ml    ______________________________________    glycophorin            10F7MN*   U.S. Pat. No. 4,752,582                                         1            D2.10     IMMUNOTECH, Marseille,                                         2                      France    CD2     6F10.3    IMMUNOTECH, Marseille,                                         3                      France    CD3     UCHT1     IMMUNOTECH, Marseille,                                         3                      France            Leu-4     Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.    CD4     Leu-3a    Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.    CD8     Leu-2a    Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.            OKT3      BioDesigns    CD14    MEM 15    Dr. Vaclav Horejsi, Institute of                                         2            MEM 18    Molecular Genetics Academy of                                         2                      Sciences of the Czech Republic,                      Praha, Czech Republic;                      Cedarlane Laboratories Hornby,                      Ontario, Canada    CD16    MEM 154*  Dr. Vaclav Horejsi, Institute of                                         2                      Molecular Genetics Academy of                                         3                      Sciences of the Czech Republic,                      Praha, Czech Republic;                      Cedarlane Laboratories Hornby,                      Ontario, Canada            3G8       IMMUNOTECH, Marseille,                      France            Leu-11a   Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.    CD19    J4.119    IMMUNOTECH, Marseille,                                         3                      France            Leu-12    Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.    CD20    MEM97     Dr. Vaclav Horejsi, Institute of                                         3                      Molecular Genetics Academy of                      Sciences of the Czech Republic,                      Praha, Czech Republic;                      Cedarlane Laboratories Hornby,                      Ontario, Canada            Leu-16    Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.    CD24    32D12*    Dr. Steinar Funderud, Institute                                         2                      for Cancer Research, Dept. of                                         3                      Immunology, Oslo, Norway            ALB9      IMMUNOTECH, Marseille,                      France    CD36    FA60152   IMMUNOTECH, Marseille,                                         3                      France            IVC7      CLB, Central Laboratory of the                      Netherlands, Red Cross Blood                      Transfusion Service    CD41    PI1.64    Kaplan, 5th International                                         3                      Workshop on Human Leukocyte                      Differentiation Antigens    CD42a   Bebl      Becton Dickinson Immuno-                                         3                      cytometry, Mountain View,                      Calif.    CD45RA  8D2.2     Craig et al. 1994, StemCell                                         1                      Technologies, Vancouver, Canada            Leu-18    Becton Dickinson Immuno-                      cytometry, Mountain View,                      Calif.    CD56    T199      IMMUNOTECH, Marseille,                                         3                      France    CD66a   CLB/gran10                      CLB, Central Laboratory of the                                         3                      Netherlands, Red Cross Blood                      Transfusion Service    CD66b   B13.9     CLB, Central Laboratory of the                                         3                      Netherlands, Red Cross Blood                                         3                      Transfusion Service            80H3      IMMUNOTECH, Marseille,                      France    ______________________________________     *preferred antibody based on performance in magnetic cell separations

                  TABLE 3    ______________________________________    Antibody Cocktails to Purify Specific Types of Lineage Committed Cells    Desired Cell    type     source of cells                        cocktail of antibodies    ______________________________________    Monocytes             Ficolled Blood                        anti-glycophorin, anti-CD2,CD3,CD56,                        CD24(CD19)             Whole Blood                        anti-glycophorin, anti-CD2,CD3,CD56,                        CD24(CD19),CD66b    B-Cells  Ficolled Blood                        anti-glycophorin, anti-CD2,CD3,CD56,                        CD14             Whole Blood                        anti-glycophorin, anti-CD2,CD3,CD56,                        CD14,CD66b    T-Cells  Ficolled Blood                        anti-glycophorin, anti-CD24(19),CD56             Whole Blood                        anti-glycophorin, anti-CD24(19),CD56,                        CD66b    CD4+ T-Cells             Ficolled Blood                        anti-glycophorin, anti-CD24(19)CD56,                        CD8             Whole Blood                        anti-glycophorin, anti-CD24(19),CD56,                        CD8,CD66b    CD8+ T-Cells             Ficolled Blood                        anti-glycophorin, anti-CD24(19),CD56,                        CD4             Whole Blood                        anti-glycophorin, anti-CD24(19),CD56,                        CD4,CD66b    NK Cells Ficolled Blood                        anti-glycophorin, anti-CD24(19),CD3             Whole Blood                        anti-glycophorin, anti-CD24(19),CD3,                        CD66b    ______________________________________

We claim:
 1. A negative selection process for enriching and recoveringhuman hematopoietic progenitor cells and human hematopoietic stem cellsin a sample containing human hematopoietic differentiated cells, humanhematopoietic progenitor cells, and human hematopoietic stem cellscomprising:(a) reacting the sample with an antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD3, CD24, CD16, and CD14 in one step under conditions so thatconjugates are formed between the antibodies and cells in the samplecontaining the antigens glycophorin A, CD3, CD24, CD16, and CD14 ontheir surfaces; (b) removing the conjugates; and, (c) recovering a cellpreparation which is enriched in human hematopoietic progenitor cellsand human hematopoietic stem cells wherein the cell preparationrecovered in step (c) has a 3 log depletion of human hematopoieticdifferentiated cells and is not labeled or coated with antibodies.
 2. Aprocess as claimed in claim 1, wherein the antibodies in the antibodycomposition are monoclonal antibodies.
 3. A process as claimed in claim2 wherein the antibodies in the antibody composition are labelled with amarker or the antibodies are conjugated to a matrix.
 4. A process asclaimed in claim 3 wherein the antibodies in the antibody compositionare labelled with biotin or a fluorochrome.
 5. A process as claimed inclaim 3 wherein the matrix is magnetic beads, a panning surface, denseparticles for density centrifugation, an adsorption column, or anadsorption membrane.
 6. A process as claimed in claim 5, wherein each ofthe monoclonal antibodies in the antibody composition is incorporated ina tetrameric antibody complex which comprises a first monoclonalantibody of a first animal species from the antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD3, CD24, CD16, and CD14, and a second monoclonal antibody of the firstanimal species which is capable of binding to at least one antigen onthe surface of a matrix, which have been conjugated to form a cyclictetramer with two monoclonal antibodies of a second animal speciesdirected against the Fc-fragments of the antibodies of the first animalspecies.
 7. A process as claimed in claim 6 wherein the antibodycomposition consists essentially of (a) antibodies specific forglycophorin A, CD3, CD24, CD16, CD14, CD66a and CD66b; or (b) antibodiesspecific for glycophorin A, CD3, CD24, CD16, CD14, CD66a, CD66b, CD56and CD2.
 8. A process for enriching and recovering human hematopoieticprogenitor cells and human hematopoietic stem cells in a samplecontaining human hematopoietic differentiated cells, human hematopoieticprogenitor cells, and human hematopoietic stem cells comprising:(a)reacting the sample with an antibody composition containing antibodiescapable of binding to the antigens glycophorin A, CD3, CD24, CD16, CD14,CD45RA, CD36, CD56, CD2, CD19, CD66a and CD66b in one step underconditions so that conjugates are formed between the antibodies andcells in the sample containing the antigens glycophorin A, CD3, CD24,CD16, CD14, CD19, CD20, CD56, CD2, CD19, CD66a and CD66b on theirsurfaces; (b) removing the conjugates; and, (c) recovering a cellpreparation which is enriched in human hematopoietic progenitor cellsand human hematopoietic stem cells wherein the cell preparationrecovered in step (c) has a 3 log depletion of human hematopoieticdifferentiated cells and is not labeled or coated with antibodies.
 9. Aprocess as claimed in claim 8, wherein the antibodies in the antibodycomposition are monoclonal antibodies.
 10. A process as claimed in claim8 wherein the antibodies in the antibody composition are labelled with amarker or the antibodies are conjugated to a matrix.
 11. A process asclaimed in claim 8 wherein the antibodies in the antibody compositionare labelled with biotin or a fluorochrome.
 12. A process as claimed inclaim 10 wherein the matrix is magnetic beads, a panning surface, denseparticles for density centrifugation, an adsorption column, or anadsorption membrane.
 13. A process as claimed in claim 8, wherein eachof the monoclonal antibodies in the antibody composition is incorporatedin a tetrameric antibody complex which comprises a first monoclonalantibody of a first animal species from the antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD3, CD24, CD16, CD14, CD45RA, CD36, CD56, CD2, CD19, CD66a and CD66b,and a second monoclonal antibody of the first animal species which iscapable of binding to at least one antigen on the surface of a matrix,which have been conjugated to form a cyclic tetramer with two monoclonalantibodies of a second animal species directed against the Fc-fragmentsof the antibodies of the first animal species.
 14. A process forenriching and recovering human hematopoietic progenitor cells and humanhematopoietic stem cells in a sample containing human hematopoieticdifferentiated cells, human hematopoietic progenitor cells, and humanhematopoietic stem cells as claimed in claim 1 wherein greater than 50%of human hematopoietic progenitor cells and human hematopoietic stemcells are recovered in step (c).
 15. A process for enriching andrecovering human hematopoietic progenitor cells and human hematopoieticstem cells in a sample containing human hematopoietic differentiatedcells, human hematopoietic progenitor cells, and human hematopoieticstem cells as claimed in claim 8 wherein greater than 50% of humanhematopoietic progenitor cells and human hematopoietic stem cells arerecovered in step (c).